Dengue viruses (DENV) are emerging, mosquito-borne Flaviviruses and the agents of dengue fever (DF) and dengue hemorrhagic fever (DHF). The NIAID has listed dengue (DEN) as a category A priority agent with respect to Biodefense and Emerging Infections research. A large body of work has demonstrated that people infected with DENV develop long-term protective immunity to the infecting serotype but not to other serotypes. During a second infection with a different serotype, the risk of severe disease is greater because cross-reactive immunity can exacerbate the disease. Efforts to develop DEN vaccines have been hampered by the dual role of immunity in protection and pathogenesis. We know very little about the antibody repertoire in people who have been infected with dengue. The main goal of this project is to use DENV3 as a model for defining the viral epitopes engaged by human antibody and to determine the functional outcome of these interactions. Our overall hypothesis is that a neutralizing antibody response is directed towards type-specific epitopes on domain III of E protein, whereas a non-neutralizing and potentially pathogenic antibody response is directed towards cross-reactive epitopes on domains I and II of E protein. In aim 1 tudies will be done to produce human monoclonal antibodies from people who have recovered from DENV nfections. In aim 2 a selected subset of human MAbs will be mapped to specific epitopes and functionally characterized for neutralizing or enhancing activity using cell culture and an animal model. The current dogma is that the main type-specific neutralization epitopes are conserved within each serotype. Our preliminary studies indicate that surface exposed regions on E protein of DENV3 are highly variable between trains. In aim 3 we will determine if the main antibody epitopes are conserved among DENV3 strains. This project takes advantage of a panel of reagents including well characterized virus strains, human samples and a DENV3 infectious clone available at UNC. The project also utilizes antibody and protein cores available within SERCEB as well as a new rodent model of DENV developed by a SERCEB investigator. The study is expected to reveal mechanisms of antibody mediated neutralization or enhancement of DENV3. This information is directly applicable to the evaluation of the safety and efficacy of candidate dengue vaccines.